Antibodies are thought to have limited protective efficacy against intracellular pathogens since they have restricted access to intracellular compartments. However, growing evidence indicates that specific antibodies play a significant role in conferring sterilizing immunity by a close and critical association with cell-mediated immune responses. The specific mechanisms of the synergistic effect of cell-mediated and humoral responses, however, remain poorly understood. In the case of Francisella tularensis, antibodies together with the cytokine IFN-? were shown to be critical in achieving rapid bacterial elimination in vivo. Furthermore, in vitro data suggest that a rapid intracellular killing of F. tularensis could be achieved if bacteria are opsonized with specific antibodies and are internalized by IFN-?-activated phagocytes. These observations lead to the hypothesis that rapid intracellular killing of intracellular bacteria by phagocytes requires the synergistic effect of IFN-? and Fc? receptor-mediated cellular events. This proposal is designed to investigate the effect of IFN-? on the fate of bacteria internalized via Fc?R by professional phagocytes and enhance the efficacy of antibodies using adjunct cytokines such as IFN-? therapy with the following specific aims: I) to determine intracellular RNS and ROS dependent bactericidal mechanisms that lead to rapid killing of opsonized F. tularensis by IFN-?- activated phagocytes, II) To determine the Fc?R- and IFN-?R-dependent signaling events leading to RNS and ROS generation by IFN-?-activated macrophages following internalization of opsonized F. tularensis, and III) to establish the trafficking of opsonized F. tularensis to various intracellular compartments of IFN-?-activated phagocytes. The results obtained will advance our understanding of interactions between humoral and cell-mediated immune responses against intracellular pathogens. The potential applications of the outcome of the study may include the design of novel prophylactic and therapeutic strategies not only against F. tularensis, but also other intracellular bacteria such as M. tuberculosis and L. pneumophila. The proposed study will also facilitate training of PI in the areas of cell and molecular biology and its application in mucosal immunobiology, areas of immediate and long term career objectives, respectively, of the candidate. The proposal and research environment at the mentor's lab complements the PI's desire to utilize basic understanding of cellular processes for therapeutic applications. There are no vaccines available against Francisella tularensis and antibody-based therapies have limited efficacy against extremely virulent form of the bacteria, the form most likely to be used by bioterrorists. We have discovered that addition of cytokine with antibodies provide improved protection and the current proposal is designed to understand the mechanism of synergy between antibodies and cytokines. These studies will result in the design of novel adjunct pharmacological agents to improve the antibody-based therapies against various bacterial pathogens.